1. Technical Field
The present invention relates generally to recombinant production of proteins. More particularly, the invention relates to methods for facilitating egress of recombinantly produced proteins from a host cell using escorts,
2. Background of the Invention
A desirable method for the production of proteins involves recombinant expression. However, commercially useful quantities of proteins can be difficult to obtain. This is particularly true with viral glycoproteins where recombinant production does not always mimic expression during viral infection. Specifically, some viral proteins are expressed at the cell surface during viral infection but are expressed intracellularly in recombinant systems. Thus, additional purification steps must be taken in order to isolate the proteins. Additionally, the proteins are susceptible to degradation by intracellular enzymes prior to purification. Such intracellular expression has been observed in herpesvirus systems with respect to glycoprotein H (gH) expression (Gompels and Minson, J. Virol. (1989) 63:4744-4755); spaete et al., Progress in Cytomegalovirus Research. (M. P. Landini, ed., 1991) pp. 133-136).
Chaperones bind newly synthesized polypeptides and appear to stabilize these polypeptides until they are assembled into their proper native structure or until they are transported to another cellular compartment, i.e., for secretion. Sambrook and Gething, Nature (1989) 342:224-225. Gompels and Minson, supra, postulate that additional virus gene products are required for transport of herpes simplex virus type 1 (HSV-1) gH through the endoplasmic reticulum-Golgi network to the cell surface. Similarly, the existence of a viral function which escorts cytomegalovirus (CMV) gH to the cell surface was proposed in Spaete et al., supra. However, CMV UL56, the homologue of herpesvirus ICP18.5 which has been implicated in the egress of viral glycoproteins to the cell surface (Pancake et al., J. Virol. (1983) 47:568-585), failed to promote movement of both truncated and full-length forms of CMV gH to the cell surface.
As with gH, cell lines transfected with the gene encoding the human immunodeficiency virus (HIV) type 1 envelope glycoprotein, gp 160, have also failed to process or export the protein. Haffar et al., J. Virol. (1990) 63:3100-3103, speculated that this was due to the absence of other viral proteins during recombinant production.
A new HSV glycoprotein, gL, has now been identified (Hutchinson et al., J. Virol. (1992) 66:2240-2250, and Hutchinson et al., Abstract, XVI International Herpesvirus Workshop, Jul. 7-12, 1991). This protein is encoded by the UL1 gene of HSV-1. Like gH, gL is not properly processed when expressed in the absence of other HSV polypeptides. However, when HSV gL and HSV gH are coexpressed, the proteins are antigenically similar to those found in infected cells and the proteins are processed and transported to the cell surface. The experimenters postulated that formation of an HSV gL/gH complex was therefore a prerequisite for the processing and transport of both molecules. However, the use of a gL/gH complex as a tool for increasing the recombinant expression of either of the proteins was not addressed.
The fibroblast growth factors (FGFs) are a family of structurally related polypeptides that regulate the growth and differentiation of a diverse number of cell types. Currently, seven distinct gene products have been identified. These include acidic and basic FGFs (Jaye et al., Science (1986) 233:541-545; Abraham et al., Science (1986) 233:545-548; Abraham et al., EMBO J (1986) 5:2523-2528), the product of the int-2 oncogene (Moore et al., EMBO J (1986) 5:919-924; Jakobovits et al., Proc. Natl. Acad. Sci. USA (1986) 83:7806-7810), a growth factor identified from Kaposi's sarcoma DNA, known as hst-1 or KS-FGF (Bovi et al., Cell (1987) 50:729-737; Taira et al., Proc. Natl. Acad. Sci. USA (1987) 84:2980-2984), FGF-5 (Zahn et al., Mol. Cell. Biol. (1988) 8:3487-3495), FGF-6 (Marics et al., Oncogene (1989) 4:335-340) and keratinocyte growth factor, KGF or FGF-7 (Finch et al., Science (1989) 245:752-755.
FGF receptors appear to mediate the effects of the various FGFs on cells. Two classes of receptors have been identified for the acidic and basic FGFs and a number of FGF receptors have now been cloned. See, e.g., Kaner et al., Science (1990) 248:1410-1413; Mansukhani et al., Proc. Natl. Acad. Sci. USA (1990) 87:4378-4382; Dionne et al., EMBO J (1990) 9:2685-2692; Mirda and Williams, Clin. Res. (1990) 38:310A; and Kiefer et al., Growth Factors (1991) 5:115-127. Kiefer et al., supra, cloned an FGF receptor from a human cell line cDNA library. The cDNA encodes a three-immunoglobulin like-domain FGF receptor and is capable of binding both acidic and basic FGFs. A soluble, extracellular domain form of this FGF receptor was produced in a baculovirus expression system and termed EC-FGF.
An FGF receptor has been implicated as a cellular receptor for HSV-1 (Kaner et al., Science (1990) 248:1410-1413). The initial HSV-1 virion attachment to cells requires an interaction with heparin like cell associated glycosaminoglycans and may be mediated by HSV envelope glycoproteins gB and gC. WuDunn and Spear, J. Virol. (1989) 63:52-58. gB and gD are essential for the secondary interactions at the cell surface that lead to virus entry into cells (Cai et al., J. Virol. (1988) 62:2596-2604; Fuller and Spear Proc. Natl. Acad. Sci. USA (1987) 84:5454-5458; Ligas and Johnson J. Virol. (1988) 62:1486-1494; Johnson et al., J. Virol. (1990) 64:2569-2576), and gH is probably also involved in the viral penetration (Fuller et al., J. Virol. (1989) 63:3435-3443). The use of an FGF receptor as an escort for increasing recombinant expression of a desired protein has not heretofore been suggested.